title: Transglutaminase-2 mediates calcium-regulated crosslinking of the Y-box 1 (YB-1) translation-regulatory protein in TGFβ1-activated myofibroblasts.
abstract: Myofibroblast differentiation is required for wound healing and accompanied by activation of smooth muscle α-actin (SMαA) gene expression. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMαA mRNA and regulates its translational activity. Activation of SMαA gene expression in human pulmonary myofibroblasts by TGFβ1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a known translation-silencer sequence in SMαA mRNA. We have determined that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent γ-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers that were distributed during SDS-PAGE over a 75-250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMαA mRNA. In human pulmonary fibroblasts, YB-1 crosslinking was inhibited by (a) anti-oxidant cystamine, (b) the reactive-oxygen antagonist, diphenyleneiodonium, (c) competitive inhibition of TG2 transamidation using the aminyl-surrogate substrate, monodansylcadaverine, and (d) transfection with small-interfering RNA specific for human TG2 mRNA. YB-1 crosslinking was partially reversible as a function of oligomer-substrate availability and TG2 enzyme concentration. Intracellular calcium accumulation and peroxidative stress in injury-activated myofibroblasts may govern SMαA mRNA translational activity during wound healing via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.
authors.affiliation: Department of Physiology and Cell Biology, The Integrated Biomedical Sciences Graduate Program, and the Ohio State Biochemistry Program, Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: William L
authors.initials: WL
authors.lastname: Willis
authors.forename: Seethalakshmi
authors.initials: S
authors.lastname: Hariharan
authors.forename: Jason J
authors.initials: JJ
authors.lastname: David
authors.forename: Arthur Roger
authors.initials: AR
authors.lastname: Strauch
mesh_terms: D000199:Actins / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D019204:GTP-Binding Proteins; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D058628:Myofibroblasts / Q000166:cytology / Q000378:metabolism; D061251:Primary Cell Culture; D014176:Protein Biosynthesis; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*; D051840:Y-Box-Binding Protein 1 / Q000378:metabolism*
chemical_list: D000199:Actins; D053773:Transforming Growth Factor beta1; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D002118:Calcium
keywords: MYOFIBROBLAST; RNA-BINDING PROTEIN; TRANSGLUTAMINASE 2; TRANSLATIONAL CONTROL; YB-1